Insights Into Peroxisome Function from the Structure of PEX3 In

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19*□S Received for publication, April 27, 2010, and in revised form, May 17, 2010 Published, JBC Papers in Press, June 16, 2010, DOI 10.1074/jbc.M110.138503 Friederike Schmidt‡1, Nora Treiber§1,2, Georg Zocher‡, Sasa Bjelic¶, Michel O. Steinmetz¶, Hubert Kalbacher‡, Thilo Stehle‡ʈ3, and Gabriele Dodt‡4 From the ‡Interfaculty Institute for Biochemistry, University of Tu¨bingen, 72076 Tu¨bingen, Germany, the §Institute for Organic Chemistry and Biochemistry, University of Freiburg, 79106 Freiburg, Germany, the ¶Laboratory of Biomolecular Research, Structural Biology, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland, and the ʈDepartment of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

The human peroxins PEX3 and PEX19 play a central role maintenance (6). Fifteen such proteins, which are named per- in peroxisomal membrane biogenesis. The membrane-an- oxins, are currently known in humans and the corresponding chored PEX3 serves as the receptor for cytosolic PEX19, genes (PEX genes) are highly conserved throughout the eukary- which in turn recognizes newly synthesized peroxisomal otic kingdom (7, 8). membrane proteins. After delivering these proteins to the All matrix proteins and most membrane proteins are Downloaded from peroxisomal membrane, PEX19 is recycled to the cytosol. The imported post-translationally into peroxisomes. The machin- molecular mechanisms underlying these processes are not ery of peroxins that mediates the import of matrix proteins well understood. Here, we report the crystal structure of the bearing a peroxisomal targeting signal is far better understood cytosolic domain of PEX3 in complex with a PEX19-derived than the machinery that mediates the recognition and import of peptide. PEX3 adopts a novel fold that is best described as a membrane proteins (9, 10). The peroxins PEX3,5 PEX16, and http://www.jbc.org/ large helical bundle. A hydrophobic groove at the membrane- PEX19 are known to be essential for peroxisomal membrane distal end of PEX3 engages the PEX19 peptide with nanomo- biogenesis as a loss of any of these proteins leads to the com- lar affinity. Mutagenesis experiments identify phenylalanine plete absence of detectable peroxisomal membrane structures 29 in PEX19 as critical for this interaction. Because key PEX3 (11). However, de novo formation of peroxisomes was observed residues involved in complex formation are highly conserved in cells deficient for each of these peroxins upon complemen- across species, the observed binding mechanism is of general tation with the wild type gene, raising an intriguing question at LIB4RI on December 18, 2018 biological relevance. about the origin of the peroxisomal membrane (11–15). The endoplasmic reticulum membrane as the obvious source was disputed for a long time as several studies indicate that this Peroxisomes are single membrane-bound organelles that process does not involve the classical coat protein I- and coat carry out a variety of metabolic processes. In addition to the protein II-dependent pathways (16–18). Recently, new evi- ␤ degradation of H2O2, the -oxidation of very long chain or dence for an involvement of the endoplasmic reticulum as a branched chain fatty acids and the synthesis of ether lipids are peroxisomal precursor has been reported in yeast (19–21) and performed in these subcellular compartments (1, 2). The bioin mammalian cells (22–24), although the details of this process genesis of peroxisomes, including their formation and prolifer- remain to be elucidated. ation, as well as the degradation of peroxisomes are highly PEX19 is a farnesylated but hydrophilic protein that is pre- dynamic processes that are adapted to metabolic needs (3). dominantly found in the cytosol, with a smaller fraction tran- Defects in peroxisome biogenesis cause a number of severe siently located at the peroxisomal membrane (12, 25). In the inherited diseases, which are collectively referred to as peroxi- cytoplasm, PEX19 can act as a chaperone for newly synthesized some biogenesis disorders (4, 5). Studies in yeast and analysis of peroxisomal membrane proteins (PMPs) by binding them dur- patients affected by these disorders have led to the identifica- ing or after translation and keeping them in an import-compe- tion of specific proteins involved in peroxisomal formation and tent form (26, 27). For the majority of PMPs, the PEX19-binding site matches the proposed membrane-targeting signal (11, * This work was supported by Deutsche Forschungsgemeinschaft Grants 28). Cargo-loaded PEX19 is directed to the peroxisomal mem- Do492/2 (to G. D.) and SFB685 (to H. K.). brane by docking to PEX3 (29). The predicted PEX3-binding The atomic coordinates and structure factors (code 3MK4) have been deposited domain of PEX19 is located within its first 56 amino acid resi- in the Protein Data Bank, Research Collaboratory for Structural Bioinformat- ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). dues, whereas the C-terminal part harbors the binding sites for □S The on-line version of this article (available at http://www.jbc.org) contains other peroxisomal membrane proteins (11, 26, 30, 31). After supplemental Figs. S1–S4 and Table S1. insertion of the PMP, PEX19 is released into the cytosol to 1 Both authors contributed equally to this work. initiate another import cycle (32). 2 Present address: Dept. of Cellular and Molecular Immunology, Max-Planck Institute of Immunobiology, 79108 Freiburg, Germany. 3 To whom correspondence may be addressed. Tel.: 49-7071-2973043; Fax: 5 The abbreviations used are: PEX, peroxisomal biogenesis factor; PMP, 49-7071-295565; E-mail: [email protected]. peroxisomal membrane protein; ITC, isothermal titration calorimetry; 4 To whom correspondence may be addressed. Tel.: 49-7071-2973349; Fax: HPLC, high pressure liquid chromatography; Bis-Tris, 2-(bis(2-hydroxy- 49-7071-295191; E-mail: [email protected]. ethyl)amino)-2-(hydroxymethyl)propane-1,3-diol.

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The peroxin PEX3 is anchored in the peroxisomal mem- 4 °C with 1 mg of tobacco etch virus protease/40 mg of protein brane via a short hydrophobic transmembrane segment while dialyzing against 1 liter of buffer A. A second Ni2ϩ col- within its N-terminal 33 residues, a region that is necessary umn removed residual uncleaved protein. Cleaved protein was and sufficient for targeting PEX3 to peroxisomes (33, 34). The then concentrated and applied to a SuperdexTM 200 10/300 cytosolic domain mediates the interaction with PEX19 (11). (GE Healthcare) size exclusion column using buffer C (50 mM PEX3 is imported into peroxisomes in a PEX19-independent Tris, 200 mM NaCl, 0.5 mM Tris-(2-carboxyethyl)phosphine, manner and hence defines a separate import pathway (26). The pH 8.0). Purity and homogeneity of the proteins were con- role of PEX16 during the import of peroxisomal membrane firmed by SDS-PAGE, native PAGE, and dynamic light scatter- proteins is less well defined, but it is thought to function as a ing. Protein folding was analyzed with circular dichroism spec- docking site for PEX3 (35). troscopy using a JASCO J-720 spectrophotometer. Protein To define the parameters that underlie the interaction of concentrations were determined by measurements of absorp- PEX3 with PEX19, we solved the structure of a soluble domain tion at 280 nm with a NanoDrop ND-1000 (PeqLab). of PEX3 in complex with a peptide corresponding to an N-ter- Peptide Synthesis—PEX19-derived peptides were prepared us- minal region of PEX19 (PEX19Pep). The soluble PEX3 domain ing solid-phase synthesis based on the N-(9-fluorenyl)methoxy- comprises residues 41–373 and contains a cysteine to serine carbonyl (Fmoc) strategy on a SyroII synthesizer (MultiSynTech, mutation at position 235 (sPEX3). In combination with affinity Witten, Germany) as described (36). Peptides were purified by measurements and mutagenesis experiments, this structure HPLC using a C18 column, resulting in a purity of 95%. Purity provides insights into the determinants of recognition of the and identity of the products were confirmed by analytical

PEX3-PEX19 complex. As residues in the contact area are HPLC, matrix-assisted laser desorption/ionization time of Downloaded from highly conserved among eukaryotes, our structure can serve as flight mass spectrometry, and electrospray ionization mass a general model for understanding the functions of PEX3 and spectrometry. PEX19 in peroxisomal biogenesis. Moreover, the structure pre- Affinity Measurements Using Isothermal Titration Calorim- sented here provides one of the first views of any interaction etry (ITC)—Affinity measurements for PEX326–373(C235S)

between two peroxins at high resolution. with full-length PEX19 were carried out in buffer C at 25 °C http://www.jbc.org/ with a VP-ITC system (Microcal). Purified PEX326–373(C235S) EXPERIMENTAL PROCEDURES was present in 8 ␮M concentration, and PEX19 was injected Protein Expression—Two human PEX3 fragments, compris- stepwise in 92 ␮M concentration. For binding studies between ing residues 26–373 and 41–373, were expressed in Escherichia sPEX3 and different PEX19-derived peptides, an ITC200 sys- coli. The corresponding DNA regions including a preceding tem (Microcal) was used. The protein was present in 17 ␮M at LIB4RI on December 18, 2018 tobacco etch virus protease cleavage site were cloned into the concentration, while the peptide fragments were injected step- ␮ vector pET32a (Novagen), which includes an N-terminal His6 wise (1 l/s) in a 5–15-fold higher concentration. ITC experi- tag. Site-directed mutagenesis (QuikChange, Stratagene) was ments with PEX19-derived peptides were performed at 25 °C in

used to generate Cys-Ser mutations at position 235 in both buffer D (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 cases. These constructs were transformed into E. coli Rosetta2 mM KCl, 0.5 mM Tris-(2-carboxyethyl)phosphine, pH 7.4). (DE3) cells and grown at 37 °C to an A600 of 0.6 before protein Binding isotherms were integrated and analyzed using Origin expression was induced with 1 mM isopropyl-␤-thiogalactopy- 7 software supplied with the instrument according to a “one ranoside. Cells were grown for an additional 16 h at 18 °C before binding site” model. harvesting. DNA coding for full-length human PEX19 with a Crystallization and Structure Determination—Purified sPEX3 preceding tobacco etch virus protease cleavage site was cloned at 2.5 mg/ml in buffer C was co-crystallized with PEX19Pep into the vector pColdI (Takara Bioscience), which includes an (residues 14–33) in a molar 1:1 ratio using the hanging drop

N-terminal His6 tag. The construct was transformed into E. coli vapor diffusion method at 20 °C. The final crystallization con- BL21 (DE3) cells. Cells were grown at 37 °C to an A600 of 0.4, at dition was optimized to 10 mM Bis-Tris, pH 5.6, 24% (w/v) poly- which point the temperature was lowered to 15 °C before pro- ethylene glycol 3350, 200 mM NaCl. Crystals appeared within tein expression was additionally induced with 1 mM isopropyl- 24 h and were flash-frozen in liquid nitrogen without adding ␤-thiogalactopyranoside. Cells were then incubated for 16 h at cryoprotectant. X-ray diffraction data were collected at beam- 15 °C. line BL14.1 (Berliner Elektronenspeicherringgesellschaft fu¨r Protein Purification—All three proteins (PEX326–373(C235S), Synchrotronstrahlung (BESSY), Berlin, Germany). They belong 41–373 1–299 PEX3 (C235S), and PEX19 ) were purified using the to space group P21 and contain one complex in the asymmetric same protocol. PEX341–373(C235S) is referred to as sPEX3. In unit. Data were processed and scaled using the XDS package each case, 10 g of cells were resuspended in 50 ml of buffer A (20 (37). Molecular replacement was carried out with PHASER (38, ␤ 26–373 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 5 mM -mer- 39) using a highly twinned structure of PEX3 (C235S) as captoethanol, pH 8.0) and lysed with an EmulsiFlex-C3 sys- the search model (40). This produced a clear solution and tem (Avestin). After centrifugation (28,000 ϫ g, 30 min, 4 °C), unambiguous difference electron density for the bound PEX19 the supernatant was loaded onto a 5-ml HisTrap HP column peptide (supplemental Fig. S3) and regions of sPEX3 that had (GE Healthcare). Protein was eluted using a linear gradient of not been included in the search model. Model building and

buffer B (20 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, 5 refinement were carried out using COOT (41) and Refmac5 mM ␤-mercaptoethanol, pH 8.0). Cleavage was carried out in a (38, 42), respectively. TLS (translation/libration/screw) groups membrane tube (Spectra/Por, Spectrumlabs) overnight at suggested by the TLS Motion Determination server (43) were

AUGUST 13, 2010•VOLUME 285•NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25411 Structure of an sPEX3-PEX19Pep Complex employed in the refinement process. According to stereochem- ical analysis within COOT, 96.9% of residues are located in favorable regions of the Ramachandran plot, whereas 3.1% are located in allowed regions. Data collection and refinement statistics are given in Table 1. Buried surface areas were calculated using CCP4 programs (38). Electrostatic surface potentials were computed with APBS (44) in PyMOL. The simulated annealing omit map was calculated in Phenix (45). The stereo view of the overall fold was displayed using Molscript (46). Coordinates and structure factors have been deposited with the RCSB Protein Data Bank (accession code 3MK4). RESULTS A PEX19-derived Peptide Binds sPEX3 with High Affinity— The interaction of the two peroxins PEX3 and PEX19 is required for the import of all peroxisomal membrane proteins. To define the structure-function relationships that underlie this interaction, we expressed and purified a soluble version of human PEX3 that comprises most of its predicted cytosolic Downloaded from domain (sPEX3) as well as a slightly longer version starting at residue 26 (PEX326–373). A solvent-exposed cysteine at position 235 was mutated to serine in both proteins to prevent non- native oxidation. Earlier crystallization trials of the larger ver-

sion of PEX3 in complex with full-length PEX19 failed. We http://www.jbc.org/ therefore pursued co-crystallization experiments of sPEX3 with a PEX19-derived peptide that spans residues 14–33 (PEX19Pep). The design was based on a predicted ␣-helix in the proposed PEX3-binding region (30) that is located within the first 56 amino acid residues of PEX19 (29). at LIB4RI on December 18, 2018 Affinity measurements using ITC demonstrate that sPEX3 Pep ϭ binds PEX19 with high affinity (Kd 330 nM, Fig. 1). The affinity of PEX326–373(C235S) for full-length PEX19 is still ϭ higher (Kd 10 nM, Fig. 1). The molar ratio was calculated to be 1:1 for both complexes (supplemental Table S1). A comparison of the ITC data shows that the enthalpy for binding PEX19Pep is 70% of the enthalpy released upon binding full-length PEX19 (supplemental Table S1). This indicates that most of the specificity of the PEX3-PEX19 interaction can be attributed to contacts with PEX19Pep. Overall Structure of sPEX3 in Complex with PEX19Pep—The FIGURE 1. ITC affinity measurements of complex formation between structure of the sPEX3-PEX19Pep complex was determined by PEX3 and PEX19. Single experiments were carried out at 25 °C. A–C, binding data for PEX326–373(C235S) with full-length PEX19 (A), sPEX3 with PEX19Pep molecular replacement at 2.42 Å resolution (Table 1 and (B), and sPEX3 with Pex19Pep F29A (C). D, integrated heat values for the differ- “Experimental Procedures”). As a search model, we used the ent ITC experiments. Closed circles, PEX326–373(C235S) and full-length PEX19; closed squares, sPEX3 and PEX19Pep; open squares, sPEX3 and Pex19Pep F29A; core structure of the previously obtained, unrefined model of see also supplemental Table S1. PEX3 comprising residues 26–373 (40). This unliganded structure could be solved to a resolution of 3.3 Å with phases ob- excellent geometry and agrees very well with the experimental ϭ tained from multiple anomalous diffraction data using seleno- data (Rfree 23.4%, Table 1). methionine-derivatized crystals, but it could not be refined due sPEX3 adopts a new fold that is composed of 10 ␣-helices and to highly twinned x-ray data (twinning factor: 0.48). However, one short 310-helix (Fig. 2 and supplemental Fig. S1). Searches components of this model led to a unique solution for the struc- for structural homologs with the programs DALI (47) and ture presented here. Unambiguous difference electron density GANGSTAϩ (48) revealed no significant matches. Although for the bound PEX19 peptide and regions of sPEX3 that had not portions of several proteins that contain multiple parallel heli- been included in the search model provide confidence for the ces can be aligned with the sPEX3 structure, the overall folds of obtained solution. The cysteine residue at position 235 is these proteins clearly differ from the sPEX3 fold, confirming located in a recessed, partially solvent-exposed area and would that sPEX3 exhibits a new fold. not be able to form intra- or intermolecular disulfide bridges in The long ␣3-helix forms the core of the structure, and the the wild type protein. Therefore, its mutation to serine is remaining helices are arranged circularly around it in five seg- unlikely to alter the sPEX3 structure. The final structure has ments (Fig. 2A). As all helical axes are aligned in roughly the

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TABLE 1 same direction, the structure can be described as a large helical Data collection and refinement statistics bundle (Fig. 2B). The bundle is about 80 Å tall and 30 Å wide. Parameter Value Interfaces between the helices are mostly hydrophobic and Data collection devoid of water molecules, providing stability to the bundle. In Beamline BL14.1, BESSY Wavelength (Å) 0.91841 contrast, the exterior of the bundle is predominantly hydro-

Space group P21 philic. The C-terminal region of sPEX3 is oriented toward the N Cell dimensions a, b, c (Å) 38.48, 65.68, 61.59 terminus and would thus face the peroxisomal membrane. Sev- ␣, ␤, ␥ (°) 90.0, 91.52, 90.0 eral of the helices in the bundle are kinked due to insertions or Resolution (Å) 25-2.42 (2.48-2.42)a a the presence of residues such as proline, which are not compat- Rmeas 5.9 (55.9) I/␴I 20.2 (3.0)a ible with a regular helical structure. These kinks are probably Completeness (%) 99.8 (99.8)a needed to allow for tight packing between helices. Although the Redundancy 3.7 (3.7)a Wilson B (Å2) 46.7 helical bundle is rigid and well defined by electron density, the Refinement positions of residues in several loop regions could not be deter- Resolution (Å) 25-2.42 a mined due to their high mobility. Regions missing from the Unique reflections 11779 (848) 146 151 220 232 245 252 a model are: Ala –Gly , Lys –Pro , Pro –Gly , Rwork/Rfree 0.194/0.234 (0.289/0.290) No. of atoms Pro301–Ser317, and Gln369–Lys373 at the C terminus. None of Protein 2249 Pep Peptide 122 these regions are close to the site of interaction with PEX19 . Water 67 Residues 14–30 of PEX19Pep are well defined by electron B-factors (Å2) Protein 39.2 density and permitted accurate model building. The C-termi- Downloaded from Peptide 58.7 nal three amino acids (Lys31–Lys33) are not visible in the elec- Water 40.8 r.m.s.b deviations tron density maps and are not included in the model. The N Bond lengths (Å) 0.01 terminus of the peptide is involved in crystal contacts that are Bond angles (°) 1.08 distant from the PEX3-binding groove. PEX19Pep forms a single a Values in parentheses are for highest-resolution shell.

␣ http://www.jbc.org/ b r.m.s., root mean square. amphipathic -helix that binds into a groove at the top of the helical bundle, opposite the N terminus of sPEX3 (Fig. 2, A and B). One side of the groove is shaped by helices ␣2 and ␣3, whereas the other side is formed by the loop that connects helices ␣4 and ␣5 and by a at LIB4RI on December 18, 2018 portion of helix ␣8. The PEX19Pep-binding Site Re- veals Three Distinct Interaction Regions within sPEX3—The contact interface is formed by surfaces in sPEX3 and PEX19Pep that are complementary in shape, producing an uninterrupted, contiguous interface that is devoid of water molecules and gaps and buries a total area of 580 Å2 from solvent. To facilitate the discussion of interactions, we have divided this interface into three contact areas along the sPEX3-binding groove (Fig. 3). The first and largest contact area (Fig. 3B and supplemental Fig. S3) is formed by sPEX3 residues located at the C-terminal end of helix ␣2 and the N-terminal region of helix ␣3. This helix-loop-helix motif of sPEX3

Pep packs tightly against the C-terminal FIGURE 2. Overall structure of sPEX3 (green) in complex with PEX19 (orange). sPEX3 folds into an Pep ␣ ␣ end of the PEX19 helix. Contacts all-helical bundle, with one central helix 3 surrounded by nine -helices and a short 310-helix. The N terminus of helix ␣1 faces toward the peroxisomal membrane. Helices were assigned with DSSP (53) and numbered are predominantly hydrophobic sequentially. A, top view of the sPEX3-PEX19Pep complex. B, side view of the sPEX3-PEX19Pep complex. Repre- sentation in A is rotated 90 degrees around a horizontal axis. C, topology of sPEX3. Helices 1–10 are represented and account for 42% of the total sur- ␣ ␣ ϩ␣ ␣ ␣ ϩ ␣ ϩ␣ as cylinders. The central helix 3 is surrounded by five helical segments ( 1 2, 4, 5 310, 6 7, face buried in the complex. A key ␣8ϩ␣9ϩ␣10) arranged in nearly parallel fashion. Regions not included in the structure are shown as dashed Pep interaction is centered at sPEX3 res- lines. D, schematic view of the secondary structure elements of sPEX3. Residues involved in PEX19 binding 104 are highlighted in bold, and residues not present in the crystal structure are shown in italics. The cysteine-serine idue Trp , which is strictly con- mutation at position 235 is boxed. served in all eukaryotic organisms

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FIGURE 4. Surface distribution of conserved amino acid residues. A, over- FIGURE 3. Interactions between sPEX3 (green) and PEX19Pep (orange). all side views of surface representation of sPEX3 differing by 180 degrees A–C, the magnified regions show details of the three major contact areas. along a vertical axis. PEX19Pep is shown as an orange ribbon. B, magnified view http://www.jbc.org/ Labels in regular font correspond to sPEX3, and labels in italics correspond to of the binding groove. PEX19Pep is displayed as a ribbon. Alignments were PEX19Pep. performed with ClustalW (54) and displayed in Jalview (55). Residues are colored according to the conservation score (supplemental Fig. S4) indicated at the lower right. Residues with a conservation score Ն 9 (9, 10ϩ, 11*) are displayed in the same color. Residues conserved in sPEX3 are colored in shades (Fig. 4 and supplemental Fig. S4A) and was previously shown to Pep be crucial for the interaction with PEX19 (49). Trp104 inserts of green, and residues conserved in PEX19 are colored in shades of orange. at LIB4RI on December 18, 2018 into a pocket formed by 4 PEX19Pep residues: Leu22, Ala25, Leu26, and Phe29. Additional hydrophobic contacts involving amino acids that were judged to be critical for the interaction sPEX3 residues Thr90, Leu93, Lys94, Lys100, Leu101, and Leu107 with sPEX3 were mutated, and the interactions between the also contribute to the interaction. peptide and sPEX3 were analyzed with ITC. Amino acid Phe29, The second contact region is formed at the loop that con- which contacts several sPEX3 residues via its hydrophobic side nects helices ␣4 and ␣5 of sPEX3 (Fig. 3A) and accounts for 21% chain (Fig. 3, B and C), was replaced with alanine. This F29A of the total buried surface area. Contacts in this region involve mutation completely abolished binding to sPEX3 (Fig. 1 and the side chains of Lys197 and Leu196 of sPEX3, which protrude supplemental Table S1). The presence of a phenylalanine at from this loop and engage residues close to the N terminus of position 29 is therefore critical for PEX3-PEX19 complex for- PEX19Pep. Lys197 forms salt bridges with Asp15 and Glu17 of the mation, consistent with the strict conservation of this residue peptide, as well as hydrophobic interactions with Leu18. Leu196 among the PEX19 sequences of eukaryotic species (Fig. 4 and packs against Leu18, Leu21, and Leu22, which all project from supplemental Fig. S4B). Secondly, PEX19Pep residue Ala25 was the same face of the PEX19Pep helix. mutated to leucine as well as tyrosine. The methyl group of The third contact region, which buries 37% of the total con- Ala25 interacts with hydrophobic residues of sPEX3 (Fig. 3, B tact area, is located at the N terminus of helix ␣8 and the loop and C). Inspection of the structure indicated that a leucine side that precedes this helix (Fig. 3C). At the center of this region lies chain might be able to form similar contacts, whereas a larger sPEX3 residue Lys324, which forms salt bridges and hydrogen tyrosine side chain would not be tolerated. Consistent with this 28 24 Pep bonds with Asp and Ser , respectively, of PEX19 . The prediction, the Kd value for the A25L mutation is 410 nM, which remaining contacts in this region are exclusively hydrophobic is only slightly lower than the affinity of wild type PEX19Pep.We and involve 2 proline residues of sPEX3. Pro321 faces toward note that a leucine is present at this position in some yeast peptide residues Leu21 and Leu22, whereas the ring of Pro327 PEX19 proteins. Not surprisingly, replacement of Ala25 with packs tightly against the Phe29 side chain of the peptide. tyrosine completely disrupted the interaction. The mutational Located near the C terminus of PEX19Pep, Phe29 also interacts data are summarized in supplemental Table S1. with the side chains of Ile326 and Asn330. Additional contacts Analysis of Surface Features and Conservation—To identify are formed with the methyl group of Ala323, which faces toward key surface features of sPEX3 and PEX19Pep, we compared the Leu21, Leu22, and Ala25 of PEX19Pep. sequences of both proteins from several organisms (supple- Mutagenesis of Contact Residues—To analyze the impact of mental Fig. S4) and displayed conserved surface residues, as mutations in PEX19Pep on sPEX3 binding, structure-guided depicted in Fig. 4. This analysis reveals that all residues lining mutagenesis experiments were performed. Select PEX19Pep the PEX19Pep-binding groove at the membrane-distal end of

25414 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 33•AUGUST 13, 2010 Structure of an sPEX3-PEX19Pep Complex sPEX3 are well conserved (Fig. 4A). The structure of the com- Several observations support our finding that the described plex identifies 2 key residues of sPEX3 that are involved in cen- binding region identified within PEX19 is indeed the main tral contacts, Trp104 and Lys324. Not surprisingly, these 2 resi- interaction surface with PEX3. Although the sPEX3-PEX19Pep dues exhibit the highest degree of conservation. A similar interaction buries only a comparatively small surface area of Pep 25 26 28 2 analysis of PEX19 residues shows that Ala , Leu , Asp , 580 Å , ITC measurements yield a high affinity of 330 nM for the 29 Pep and Phe are most highly conserved (Fig. 4B). Again, these sPEX3-PEX19 complex. The Kd value for the interaction 26–373 residues all play central roles in complex formation (Fig. 3 and between PEX3 (C235S) and PEX19 is 10 nM, indicating a supplemental Table S1). An interesting finding is that the 33-fold higher affinity for full-length PEX19. This value is con- C-terminal region of PEX19Pep (residues 25–29) is more consistent with previous surface plasmon resonance studies result- served than residues that lie closer to the N terminus of the ing in a Kd value in the low nanomolar range (3.4 nM) and a 1:1 peptide (residues 18–24). This indicates that contacts formed molar ratio between PEX3 and PEX19 (49). The high affinity for by the C-terminal portion of PEX19Pep, and especially residues PEX19Pep can be attributed to an interacting surface that is Leu26 and Phe29, are most important for a productive interac- complementary in shape, devoid of water molecules, and pre- tion and for conferring specificity. dominantly hydrophobic. The higher affinity for full-length Although most of the remaining sPEX3 surface is quite vari- PEX19 could be due to additional residues within the PEX19 N able, our analysis reveals a second cluster of highly conserved terminus, which were not included in the synthesized peptide residues at the base of the molecule, near the N-terminal helix but which contribute to binding at the identified interaction ␣1. This cluster includes an unusual number of surface-ex- site. However, because both termini of the PEX19Pep helix point 49 67 72 135 posed large, hydrophobic residues (Ile , Met , Met , Ile , away from sPEX3 and because the sPEX3 structure lacks addi- Downloaded from and Ile140). In vivo, this region would be located close to the tional conserved residues at the top of the bundle, we consider peroxisomal membrane. It is tempting to speculate that it forms this possibility unlikely. The observation that the enthalpy for the site of interaction with a second region of PEX19 or with PEX19Pep binding is 70% of the enthalpy for the full-length other peroxisomal proteins. protein indicates that PEX19Pep contributes significantly to the

Analysis of surface charges of sPEX3 (supplemental Fig. S2A) interaction. However, the difference in Kd also suggests that http://www.jbc.org/ reveals a polar surface with many small positively and nega- other residues of the full-length PEX19 protein are likely in- tively charged patches and two noticeably larger ones. The volved in contact formation. The conserved region at the PEX19Pep-binding groove exhibits a strong positive potential, base of sPEX3 is a possible candidate for additional interac- consistent with several basic residues (Lys94, Lys100, Lys197, and tions with full-length PEX19. A second binding site within Lys324) that line the groove and participate in PEX19Pep bind- residues 124–140 of PEX19 has been proposed previously at LIB4RI on December 18, 2018 ing. In contrast, the PEX19-derived peptide exhibits a highly (30). However, this region is unlikely to interact with PEX3 negative surface potential due to exposed aspartic and glutamic as our ITC measurements clearly show that a PEX19-derived acids (supplemental Fig. S2B). A less conserved region at the peptide corresponding to these amino acids does not bind base of sPEX3 exhibits a strong negative potential, which is due sPEX3 (supplemental Table S1). to an accumulation of acidic residues (Asp257, Glu266, Asp269, The functional relevance of the observed interaction be- Glu272, and Asp275). Among these residues, Glu266, Glu272, and tween sPEX3 and PEX19Pep is further supported by the pres- Asp275 are highly conserved in all eukaryotic species except ence ofhighly conserved residues in the peptide-binding groove. A yeasts. These features may indicate functional interactions conserved region including PEX3 residues Lys100 to Arg114 was of these residues with other proteins during peroxisomal predicted to contribute to PEX19 binding (49). Our structure biogenesis. shows that many of these residues indeed form key contacts with PEX19Pep. Residues Lys100, Leu101, Trp104, and Leu107 are DISCUSSION all part of the largest binding region (Fig. 3B). Trp104 can even PEX3 has been reported to participate in different processes be considered a central residue as it interacts with several during peroxisomal biogenesis. In this study, we sought to hydrophobic PEX19Pep side chains, including Phe29. Further- define its structural features, as well as the specificity of its more, the tryptophan helps to orient side chains of additional interaction with PEX19. Experiments performed in several lab- sPEX3 residues (Leu93 and Lys100) to form contacts with oratories have identified the N-terminal 56 amino acids of PEX19Pep. Consistent with the central role of Trp104 in complex PEX19 as necessary and sufficient for the interaction with PEX3 formation, it was reported that its replacement with alanine and thus for docking PEX19 to the peroxisomal membrane (26, reduces the affinity of PEX3 for PEX19 significantly. Moreover, 29–31). Based on these studies, we have designed and synthe- the W104A mutant of PEX3 was unable to restore peroxisomes sized a peptide that comprises the central part of this region of in a PEX3-deficient cell line (49). Our surface analysis reveals human PEX19 and determined the structure of the cytosolic that the PEX19Pep residues involved in sPEX3 recognition are domain of human PEX3 in complex with this peptide. sPEX3 also highly conserved throughout eukaryotic species. The folds into an elongated helical bundle that has no known struc- importance of these residues is emphasized by mutagenesis tural homologs. Structural and functional analyses identified a experiments and affinity measurements, identifying Phe29 as single groove in sPEX3 as a high affinity binding site for PEX19. crucial for the formation of a PEX3-PEX19 complex (Fig. 1 and This region is highly conserved across eukaryotic species. Addi- supplemental Table S1). tional regions of surface conservation of sPEX3 indicate contact The observed contacts between sPEX3 and PEX19Pep are in points for other molecules involved in peroxisomal biogenesis. line with some, but not all, earlier studies that have sought to

AUGUST 13, 2010•VOLUME 285•NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25415 Structure of an sPEX3-PEX19Pep Complex identify PEX3 residues involved in the interaction with PEX19. adjacent to the membrane and its subsequent insertion. Fang et al. (29) proposed that the highly conserved PEX3 resi- Although the PEX3-PEX19 complex formation is clearly ex- dues 120–136 interact with PEX19 as this segment colocalizes plained by the structure presented here, the membrane posi- with a nuclear localization sequence-tagged version of PEX19 tioning and insertion of PMPs remain speculative. It is known to the nucleus. Furthermore, mutations in this region disrupted that PEX19 is composed of a flexible N-terminal domain and a interactions with PEX19 and the ability of PEX3 to complement compact, farnesylated C-terminal domain (51), with the farne- PEX3-deficient human cells. However, these PEX3 residues are sylation being crucial for correct PMP targeting to peroxisomes in fact distant from the PEX19-binding site. They are located (52). It is likely that upon PEX19 binding to PEX3, the cargo- within helix ␣3, at the center of the protein, and are part of the loaded C terminus of PEX19 is oriented close toward the per- solvent-inaccessible, hydrophobic core. These residues are oxisomal membrane. This could involve either additional inter- therefore not able to interact with PEX19 in a physiological actions with PEX3, perhaps mediated by the farnesyl group, or setting. The observed colocalization is probably due to unspe- a direct insertion of the prenyl anchor into the peroxisomal cific binding of PEX19 to hydrophobic PEX3 regions. As sPEX3 membrane. The mechanism of PMP insertion could also residues 120–136 help to stabilize the protein fold, mutations involve PEX16. The release of cargo from PEX19 could be trig- in this region destabilize the entire ␣-helical bundle and are gered by a conformational change in PEX19 or by binding of thus likely to abolish proper function of PEX3. PMPs to PEX3. This might be sufficient for dissociation of the The surface analysis of sPEX3 reveals a mostly hydrophilic PEX3-PEX19 complex. One other possibility is that the affinity surface and strongly argues against the insertion of PEX3 into for PEX3 is reduced once PEX19 has unloaded its cargo. In the peroxisomal membrane. Instead, our structure supports a support of this, it has been shown that the affinity of PEX3 for Downloaded from model in which PEX3 is anchored to peroxisomes with a single PEX19 carrying green fluorescent protein (GFP)-PMP24 is sequence located at the N terminus but has no other direct higher than for cargo-free PEX19 (27). Thus, cargo-loaded contact with the lipid bilayer. An earlier study reported an PEX19 could displace cargo-free PEX19 from PEX3 at the per- interaction of the cytosolic domain of PEX3 with membrane oxisomal membrane to initiate a new insertion cycle.

lipids, based on the observation that PEX3 forms high molecu- In conclusion, our structure provides one of the first views of http://www.jbc.org/ lar mass aggregates in the presence of mild detergents (50). This a complex between two peroxins at high resolution. It reveals lipid binding property was assigned to hydrophobic residues in essential structural features of sPEX3 and establishes a platform several predicted amphipathic helices. However, our structure for understanding the parameters that guide its interactions shows that these hydrophobic residues are in fact part of the with PEX19. Moreover, surface conservation analysis of sPEX3 solvent-inaccessible core of the protein and are thus not avail- provides a basis for potential interaction with other molecules at LIB4RI on December 18, 2018 able for interactions with lipids. As the lipid binding activity can and for the general role of PEX3 in peroxisomal membrane be abolished by the addition of recombinant PEX19, a compet- biogenesis. ing interaction between PEX3 and lipids on one side and PEX3 and PEX19 on the other side has been suggested (50). We note Acknowledgments—We thank the beamline staff of BESSY (Berlin, that the borders of the PEX19Pep-binding groove, as well as a Germany) for support during data collection. The structure determi- region just beyond the groove, exhibit a basic character due to nation of PEX3 was initiated in the laboratory of Prof. Dr. Georg E. the presence of highly conserved positively charged amino Schulz (University of Freiburg, Germany). ITC measurements of acids (Arg96, Lys100, and Lys324) (Fig. 4 and supplemen- PEX3 with full-length PEX19 were carried out by Dr. Carsten tal Fig. S4). Thus, we cannot rule out the possibility that these 3 Kintscher at the Max-Planck Institute for Developmental Biology in residues form favorable charge-charge interactions with mem- Tu¨bingen, Germany. brane phospholipids, which are abolished once PEX19 engages PEX3 with high affinity. Although such interactions with phos- REFERENCES pholipids explain the observed lipid binding capacity of the cytosolic domain of PEX3 in vitro, they are not likely to be 1. Wanders, R. J., and Waterham, H. R. (2006) Biochim. Biophys. Acta 1763, 1707–1720 relevant under physiological conditions. 2. Schrader, M., and Fahimi, H. D. (2008) Histochem. Cell Biol. 129, 421–440 It is known that PEX19 is recycled back to the cytosol after 3. Huybrechts, S. J., Van Veldhoven, P. P., Brees, C., Mannaerts, G. P., Los, cargo release into the peroxisomal membrane (32). Thus, the G. V., and Fransen, M. (2009) Traffic 10, 1722–1733 high affinity interaction between PEX3 and PEX19 must be dis- 4. Weller, S., Gould, S. J., and Valle, D. (2003) Annu. Rev. Genomics Hum. rupted to start a new insertion cycle. The structure of the com- Genet. 4, 165–211 plex does not offer any plausible scenarios for how this might 5. Steinberg, S. J., Dodt, G., Raymond, G. V., Braverman, N. E., Moser, A. B., and Moser, H. W. (2006) Biochim. Biophys. Acta 1763, 1733–1748 occur. Subtle changes in pH are known to drive the association 6. Platta, H. W., and Erdmann, R. (2007) Trends Cell Biol. 17, 474–484 and dissociation of other large complexes. However, as the 7. Kiel, J. A., Veenhuis, M., and van der Klei, I. J. (2006) Traffic 7, 1291–1303 sPEX3-PEX19Pep interface is largely hydrophobic, changes in 8. Tabak, H. F., Hoepfner, D., Zand, A., Geuze, H. J., Braakman, I., and pH are unlikely to affect its stability. Instead, we consider it Huynen, M. A. (2006) Biochim. Biophys. Acta 1763, 1647–1654 more likely that other peroxins might play a role in this process. 9. Girzalsky, W., Saffian, D., and Erdmann, R. (2010) Biochim. Biophys. Acta Our results can serve as a model of the general mechanism of 1803, 724–731 10. Alencastre, I. S., Rodrigues, T. A., Grou, C. P., Fransen, M., Sa´-Miranda, C., peroxisomal membrane protein import. Membrane insertion and Azevedo, J. E. (2009) J. Biol. Chem. 284, 27243–27251 of PMPs has to involve the recruitment of PMP-loaded PEX19 11. Fujiki, Y., Matsuzono, Y., Matsuzaki, T., and Fransen, M. (2006) Biochim. to the peroxisome followed by positioning of the PMP directly Biophys. Acta. 1763, 1639–1646

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AUGUST 13, 2010•VOLUME 285•NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25417 Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19 Friederike Schmidt, Nora Treiber, Georg Zocher, Sasa Bjelic, Michel O. Steinmetz, Hubert Kalbacher, Thilo Stehle and Gabriele Dodt J. Biol. Chem. 2010, 285:25410-25417. doi: 10.1074/jbc.M110.138503 originally published online June 16, 2010

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